Portions of this work were presented at the 19th Annual Meeting of the American Society for Bone and Mineral Research, Cincinnati, Ohio, U.S.A., 1997.
The Vitamin D Receptor Gene Start Codon Polymorphism: A Functional Analysis of FokI Variants†
Article first published online: 1 NOV 1998
Copyright © 1998 ASBMR
Journal of Bone and Mineral Research
Volume 13, Issue 11, pages 1691–1699, November 1998
How to Cite
Gross, C., Krishnan, A. V., Malloy, P. J., Eccleshall, T. R., Zhao, X.-Y. and Feldman, D. (1998), The Vitamin D Receptor Gene Start Codon Polymorphism: A Functional Analysis of FokI Variants. J Bone Miner Res, 13: 1691–1699. doi: 10.1359/jbmr.1918.104.22.1681
- Issue published online: 4 DEC 2009
- Article first published online: 1 NOV 1998
- Manuscript Accepted: 27 JUL 1998
- Manuscript Revised: 22 JUN 1998
- Manuscript Received: 6 MAR 1998
The vitamin D receptor (VDR) gene contains a start codon polymorphism (SCP) which is three codons upstream of a second start site (ATG). The SCP genotype can be determined with the restriction enzyme FokI, where “f” indicates the presence of the restriction site and the first ATG, while “F” indicates its absence. Recent evidence suggests that the ff genotype is correlated with lower bone mineral density (BMD) in some populations. The SCP results in alternate VDRs that differ structurally, with the F variant (F-VDR) being three amino acids shorter than the f variant (f-VDR). To determine whether there are functional differences between the f-VDR and the F-VDR, we studied the two VDR forms expressed in COS-7 cells. The proteins were distinguishable from one another on Western blots by their different mobilities, confirming the larger size of f-VDR. Ligand binding studies showed no significant differences between the affinities of the two VDR forms for [3H]-1,25-dihydroxyvitamin D3 ([3H]-1,25(OH)2D3) (Kd = 131 ± 78 pM, f-VDR; Kd = 237 ± 190 pM, F-VDR; p = 0.24); however, a 2-fold difference in affinity can not be discriminated by this method. There were no differences in the abilities of the two receptor forms to bind DNA as determined by electrophoretic mobility shift assays. The ability of the two VDR forms to transactivate target genes was investigated using three different vitamin D responsive luciferase reporter constructs: 24-hydroxylase, osteocalcin, and osteopontin. In these transactivation experiments, 1,25(OH)2D3 dose-response (0.1–10 nM) curves revealed that the ED50 values for transactivation were indistinguishable between the two VDR forms. Additionally, cultured human fibroblasts with FF,Ff, and ff genotypes had similar sensitivity to 1,25(OH)2D3 with respect to the induction of 24-hydroxylase mRNA. In summary, we were unable to detect significant differences in ligand affinity, DNA binding, or transactivation activity between f-VDR and F-VDR forms. We must emphasize, however, that the sensitivity of the methods used limits our ability to detect minor differences in VDR affinity and function. In conclusion, we cannot define a mechanism whereby the SCP in the VDR might contribute to population differences in BMD.
THE NUCLEOTIDE SEQUENCE of the human vitamin D receptor (VDR) gene contains two potential translation initiation (ATG or start) sites.1 A polymorphism has been described in the first start codon which changes the nucleotide sequence to ACG.2 Alleles with the ATG polymorphism would be predicted to start VDR translation at the first ATG, and alleles with the ACG polymorphism would be expected to initiate translation at the ATG three codons downstream. Thus, alternate start codon polymorphism (SCP) alleles would give rise to VDR proteins that differ in length by three amino acids.
Because of the central role of the VDR in mineral homeostasis and the possibility that this polymorphism alters VDR function, we developed a restriction fragment length polymorphism assay using the FokI enzyme.3 In this assay, “f” indicates the presence of the FokI restriction site and “F” indicates its absence. Thus, the f allele contains both ATGs, whereas the F allele has only the second ATG, and thus predicts a shorter VDR protein. We3,4 and others5 have shown that the SCP correlates with bone mineral density (BMD) such that individuals homozygous for both ATGs (ff) have lower BMD than individuals homozygous for the absence of the first ATG (FF). Heterozygotes (Ff) have intermediate BMD. However, this association is not present in all populations.4,6
In a preliminary report, Sturzenbecker et al.7 have shown that the alternate forms of the VDR function similarly in transactivation assays using 24-hydroxylase and osteocalcin reporter genes. In contrast, Arai et al.5 detected a difference between the two VDR forms using a similar assay system. They showed that the longer form of the VDR (comparable to f) functioned 40% less well than the shorter form (comparable to F). In the present study, we have further investigated these two VDR forms (f-VDR and F-VDR), examining multiple steps involved in the vitamin D action pathway. We compared the two receptor forms with respect to binding affinity for [3H]-1,25-dihydroxyvitamin D3([3H]-1,25(OH)2D3), the ability to dimerize with the retinoid X receptor (RXR) and interact with DNA in electrophoretic mobility shift assays (EMSA), as well as the ability to transactivate three different vitamin D responsive luciferase reporter gene constructs transfected into COS-7 cells. We have also investigated the ability of 1,25(OH)2D3 to induce endogenous 24-hydroxylase mRNA in normal human fibroblasts of FF, Ff, and ff genotypes.
MATERIALS AND METHODS
1α,25(OH)2[26,27 methyl3H]vitamin D3 (specific activity 174 Ci/mmol), [α-32P]dCTP (3000 Ci/mmol), and [γ-32P]ATP (3000 Ci/mmol) were obtained from Amersham Chemical Co. (Arlington Heights, IL, U.S.A.). Nonradioactive 1α,25(OH)2D3 was the generous gift of Dr. M. Uskokovic (Hoffmann La-Roche Co., Nutley, NJ, U.S.A.). Culture media were obtained from Mediatech (Herndon, VA, U.S.A.). Aprotinin, pepstatin, soybean trypsin inhibitor, and the random primed DNA labeling kit were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN, U.S.A.). Restriction enzymes and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA, U.S.A.). Polymerase chain reaction (PCR) reagents were purchased from GIBCO BRL (Gaithersburg, MD, U.S.A.), Stratagene (San Diego, CA, U.S.A.), and Invitrogen (San Diego, CA, U.S.A.). All other reagents, except where indicated, were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.).
DNA was isolated from whole blood or cultured cells using the QIAamp kit (Qiagen, Santa Clarita, CA, U.S.A.). The PCR conditions for amplifying exon 2 of the VDR gene and genotyping at the SCP locus have been described previously.3,4,6 Amplification was carried out using the primers VDR2a: 5′-AGCTGGCCCTGGCACTGACTCTGCTCT-3′ and VDR2b: 5′-ATGGAAACACCTTGCTTCTTCTCCCTC-3′. PCR products were digested with FokI and then electrophoresed through 2% agarose gels containing ethidium bromide. Genotypes were scored as FF homozygotes, Ff heterozygotes, or ff homozygotes according to the digestion pattern.
COS-7, MCF-7, and T47-D cells were obtained from American Type Culture Collection (Rockville, MD, U.S.A.). Strains of fibroblasts were cultured from skin biopsies of donors using previously described methods8 or were provided by Dr. Walter Horton. The protocol was approved by the institutional review board for human subjects at Stanford University. Cells were incubated at 37°C under a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM; human skin fibroblasts and COS-7) or in RPMI 1640 (MCF-7 and T47-D) with fetal bovine serum (FBS) or iron-supplemented calf serum (Fe-CS; HyClone Laboratories, Logan, UT, U.S.A.).
Construction of VDR expression vectors
Human VDR cDNA was amplified from the pGEM-hVDR construct1 with 5′ primers engineered with either the ATG or ACG polymorphic sequence and an upstream XhoI restriction sequence and a 3′ primer directed to the 3′ UTR (FUS-2). f-VDR: 5′-CCGCTCGAGATGGAGGCAATGGCGGCCAGCACTTCCCTGCCT-3′; F-VDR: 5′-CCGCTCGAGACGGAGGCAATGGCGGCCAGCACTTCCCTGCCT-3′; FUS-2: 5′-GAGGTCGACTAGTCAGGAGATCTCATT-3′. After 30 cycles of amplification at 95°C for 20 s, 60°C for 20 s, and 72°C for 60 s, followed by 72°C for 2 minutes, the PCR products were gel purified. The PCR products were digested with XhoI and SalI and then subcloned into the XhoI site in pBluescript KSII+ (Stratagene, San Diego, CA, U.S.A.). The f-VDR and F-VDR cDNAs were then subcloned into the BamHI and XhoI sites of pEUK-C1 (Clontech, Palo Alto, CA, U.S.A.) in the proper orientation to allow for expression.
Cytosol extracts of COS-7 cells transfected with the f-VDR or F-VDR expression plasmids were prepared with KTEDM buffer (300 mM KCl, 10 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 1 mM dithiothreitol, and 10 mM sodium molybdate) containing a protease inhibitor cocktail (10 μg/ml soybean trypsin inhibitor, 1 μg/ml leupeptin, 2 μg/ml pepstatin, 1 μg/ml aprotinin). Cystosol extracts were diluted to 0.1 mg/ml protein with KTEDM and adjusted to 1 mg/ml with gelatin. [3H]-1,25(OH)2D3 binding was performed as described previously.8,9
COS-7 cells were seeded in 6-well plates at a density of 1.5 × 105 cells per well and allowed to attach overnight at 37°C in DMEM with 10% FBS. The following day, the cells were transfected with plasmid DNA using 5 μl of LipofectAMINE (GIBCO BRL) in OptiMEM (GIBCO) according to the instructions of the manufacturer. Cells were tranfsected with VDR expression plasmids (0.5 μg/well, except where indicated) and one of three vitamin D responsive reporter constructs (1 μg/well): the rat 24-hydroxylase promoter-luciferase (24OHase-Luc),10 the human osteocalcin vitamin D response element (VDRE)-luciferase (OC-Luc), or the mouse osteopontin VDRE-luciferase (OP-Luc) reporter gene constructs (generously supplied by Dr. J.W. Pike). The OC-Luc and OP-Luc reporters contain their respective VDREs in a MMTV LTR-Luciferase construct. The pRL-SV40 renilla luciferase expression plasmid (0.02 μg/well; Promega, Madison, WI, U.S.A.) was included in the cotransfections to control for transfection efficiency. After 7 h of incubation with the DNA-lipid mixture in OptiMEM (GIBCO BRL), the medium was replaced with DMEM containing 1% charcoal stripped FBS and various concentrations of 1,25(OH)2D3 or vehicle (ethanol 0.1%). The cells were incubated at 37°C for 18 h. Cells were harvested and lysates prepared in Passive Lysis Buffer (Promega). Luciferase activity was determined using the Dual Luciferase Reporter System Assay (Promega) in a Turner Designs 20/20 luminometer (Sunnyvale, CA, U.S.A.). All experiments were done in triplicate. The coefficients of variation in these assays were 24OHase-Luc, 6.0 ± 2.6%; OC-Luc, 5.6 ± 3.7%; and OP-Luc, 7.8 ± 4.0%.
Western blot analysis
Western blot analysis was carried out as previously described.11 The 9A7γ rat antichick VDR monoclonal antibody (MAb) was used as the primary antibody.
Electrophoretic mobility shift assay
EMSAs were performed as previously described.12,13 Briefly, 2 μl (7 fmol) of cytosol extracts from COS-7 cells transfected with VDR expression plasmids were incubated with RXRα, and a [32P]-labeled double-stranded oligonucleotide corresponding to the human osteopontin VDRE (OP-VDRE) nucleotide sequence13,14 in the presence of 100 nM 1,25(OH)2D3 or ethanol vehicle control. Increasing concentrations of unlabeled OP-VDRE were added as a competitor in some reactions. In some samples the VDR MAb, 9A7γ, was added prior to the addition of labeled OP-VDRE.
RNA isolation and Northern blot analysis
Northern analysis was done as previously described.8,11,15 At subconfluence, cells were incubated in the presence of 1,25(OH)2D3 or vehicle (ethanol 0.1%) in DMEM with 1% Fe-CS plus antibiotics for 6 h. Northern analysis was carried out with a [32P]-labeled DNA fragment of the rat 25(OH)D3 24-hydroxylase gene.16 To control for RNA sample loading and transfer, Northern blots were also hybridized with a [32P]-labeled fragment of the cDNA for the human ribosomal protein gene L7, which has been shown not to change with the cell cycle, serum stimulation, or physiological state.11,17 Quantitation of bands of interest were determined by laser densitometry on a densitometer (Molecular Devices, Menlo Park, CA, U.S.A.).
Data are presented as mean ± SD. Statistical analysis was done by t-test or analysis of variance using Statview 4.5 software (Abacus Concepts, Berkeley, CA, U.S.A.). A p value of ≤ 0.05 was considered significant.
Western analysis of VDR
To confirm the predicted size difference between the f-VDR and the three amino acid shorter F-VDR, we performed Western blot analyes on a variety of cell types. F-VDR and f-VDR forms were overexpressed in COS-7 cells, and cytosol extracts were analyzed with the rat antichick VDR Mab, 9A7γ (Fig. 1A, lanes labeled C). The two VDR forms differ in size by ∼400 Da as predicted by their cDNA sequences. Cytosol extracts from homozygous FF and ff fibroblasts similarly demonstrate this size difference. Moreover, Ff heterozygous fibroblasts (Fig. 1A) showed a characteristic VDR doublet, indicating that both the f and F alleles are expressed in these cells. Figure 1B demonstrates that human breast cancer cells, which are heterozygous at the f allele (T47D), also express both f and F alleles. MCF-7 breast cancer cells are homozygous FF and express only the F-VDR as expected. There was no detectable difference in the abundance of F-VDR and f-VDR in Ff heterozygous cells in multiple blots, suggesting equivalent expression of VDR alleles. Interestingly, up-regulation of both F-VDR and f-VDR in response to 1,25(OH)2D3 is apparent in T47D (Fig. 1B). Homologous up-regulation of the VDR in breast cancer cell lines has been previously described.18–20
F-VDR and f-VDR forms were expressed in COS-7 cells, and cytosolic extracts were made to study the affinity of the receptor variants for 1,25(OH)2D3. Figure 2 shows representative Scatchard plots of [3H]1,25(OH)2D3 binding at 0°C in F-VDR and f-VDR expressed in COS-7 cells. The mean dissociation constant values (Kd) were not significantly different; the Kd for f-VDR was 131 ± 78 pM and for F-VDR 237 ± 190 pM (p = 0.24, n = 3). Similarly, when binding was carried out at 25°C, there were also no significant differences in Kd (data not shown). The relative amounts of f-VDR and F-VDR (Nmax) varied in ligand binding experiments due to different transfection efficiencies.
To determine if the f-VDR and F-VDR forms had similar affinities for DNA, we investigated the ability of the two receptor forms to bind the human OP-VDRE by EMSA. The EMSA shown in Fig. 3 demonstrates the ability of both VDR forms to dimerize with RXR and bind with the OP-VDRE in a specific manner. As shown in Fig. 3, addition of increasing concentrations of unlabeled OP-VDRE reduced the band shift equally in both VDR forms, indicating that the two receptor forms have similar affinities for DNA.
Transactivation of reporter genes
The ability of f-VDR and F-VDR forms to transactivate target genes was examined in COS-7 cells cotransfected with either f- or F-VDR expression plasmids and one of three vitamin D responsive reporter gene constructs. Transfected cells were treated with a range of 1,25(OH)2D3 concentrations (0.1–10 nM). As shown in Fig. 4, there were no differences in the dose-response curves between f-VDR and F-VDR using the 24OHase-Luc (Fig. 4A), OC-Luc (Fig. 4B), and OP-Luc (Fig. 4C) reporters, respectively. The data are corrected for transfection efficiency by assay of renilla luciferase, which was expressed from pRL-SV40 included in the transfections (see Materials and Methods). The ED50 values for the f-VDR and F-VDR are 0.4 versus 0.3 nM for the 24OHase-Luc reporter, 1.5 versus 1.4 for the OC-Luc reporter, and 1.2 versus 1.4 for the OP-Luc reporter, respectively. Western blot analyses demonstrated similar expression levels of f-VDR and F-VDR in these transactivation assays (Fig. 4, right panels).
Arai et al.5 found that the f-VDR was less active than the F-VDR in transactivating a 24OHase-Luc reporter at 50 nM 1,25(OH)2D3. In contrast, our initial dose-response experiments failed to find differences in transactivation activities between the two receptor forms. We therefore used the same 50 nM dose of 1,25(OH)2D3 as was employed in the experiments of Arai et al.5 At this concentration, we were unable to detect differences in transactivation activities between f-VDR and F-VDR on the 24OHase-Luc reporter. There was a 10.07 ± 1.08-fold induction of 24OHase-Luc for f-VDR from a baseline activity of 0.22 ± 0.12 light units versus 9.92 ± 0.78-fold induction for F-VDR from a baseline activity of 0.21 ± 0.09 light units, p = 0.54. Western blot analysis of lysates from transfected cells revealed similar levels of VDR expression (data not shown). The reporter we used contains nucleotides −1399 to +76 of the rat 24-hydroxylase gene promoter.10 This differs from the reporter construct used by Arai et al.,5 which contains only nucleotides −291 to +9 of the rat 24-Hydroxylase promoter.
Because the abundance of VDR in the transfected COS-7 cell system is ∼500–2000 fmol/mg of protein, these findings may not reflect VDR function in normal intact cells where the VDR content is only 20–50 fmol/mg of protein. Thus, we titrated the amount of transfected VDR expression vector to investigate whether lower levels of VDR would bring out subtle differences in transactivation activity between f-VDR and F-VDR. Figure 5 demonstrates that lower amounts of transfected VDR expression plasmid (0.025–0.5 μg/well) failed to reveal differences in transactivation activity on the 24OHase-Luc reporter.
24-Hydroxylase mRNA induction in fibroblasts
We also investigated the ability of the two VDR forms to induce the endogenous 24OHase mRNA in human dermal fibroblasts of the ff, Ff, and FF genotype. Subconfluent cultures of fibroblasts were treated with increasing concentrations of 1,25(OH)2D3 (0.1–10 nM) or vehicle for 6 h. Total RNA was isolated, and 24OHase mRNA levels were examined by Northern blotting. As shown by Northern analysis in Fig. 6A, 24OHase mRNA levels increased with increasing 1,25(OH)2D3 concentrations in all three cell types. After correction for gel loading, using L7 RNA, no substantial differences in cellular sensitivity to 1,25(OH)2D3 between genotypes was noted. The ED50 values determined from densitometric analysis for 24OHase mRNA induction were ff, 0.9 nM; Ff, 1.3 nM; FF 0.6 nM (Fig. 6B).
The observation that polymorphisms in the VDR gene are associated with variations in BMD has engendered a large body of research over a short period of time. The most commonly studied polymorphisms, which are located in the 3′ end of the gene and are defined by the restriction enzymes BsmI, ApaI, TaqI, do not produce changes in the predicted amino acid sequence of the VDR.21,22 As a result, it has been difficult to rationalize how these polymorphisms might lead to differences in BMD. Indeed, several studies have been unable to elucidate the molecular mechanism that underlies the association of these VDR polymorphisms with variations in BMD.15,22–25 The SCP of the VDR has also been linked with differences in BMD. This association has been found in some populations3–5 but not in others.4,6 The SCP leads to the production of VDR proteins that differ in length by three amino acids, which may result in differential VDR function. Thus, the VDR SCP provides a potential molecular mechanism to explain the observed association of this polymorphism with differences in BMD among some populations.
Our Western blot analysis shows that the f-VDR and F-VDR differ in size as predicted by their cDNA sequences. These results demonstrate that both the first and second ATG can act as translation initiation sites, and that only the most upstream ATG is used when both are present (f alleles). In addition, we demonstrate that cells heterozygous for the SCP express both VDR forms. Furthermore, the abundance of the two VDR forms in heterozygous cells appears equal.
The structural differences between the two VDR forms suggested the potential for differences in ligand and/or DNA binding. However, in [3H]1,25(OH)2D3 binding and EMSA studies, we observed no significant differences in hormone and DNA binding affinities between the two VDR forms. Although there was a statistically nonsignificant trend for a lower Kd for f-VDR compared with F-VDR, the power of our ligand-binding studies limited our ability to detect less than a 2-fold difference in VDR affinity for 1,25(OH)2D3. The EMSA data also suggest that the interactions between RXR and the two VDR forms are similar.
Previous studies by Arai et al.5 have shown that the f-VDR functions ∼40% less well in transactivation experiments than the F-VDR. This apparent decrease in function of the f-VDR would appear to provide a molecular mechanism to explain why individuals with the ff genotype have lower BMD.3–5 In contrast, our results do not support the findings of Arai et al.5 We found no significant differences between the transactivation activities of f-VDR and F-VDR using a 24OHase-Luc reporter. These discrepant results may be due to differences in the reporter constructs employed in the two studies. As mentioned above, we used a reporter construct that contains a large portion of the promoter region from the rat 24-hydroxylase gene (nucleotides −1399 to +76),10,26 whereas Arai et al.5 used a truncated portion of the rat promoter that includes only nucleotides −291 to +9. These differences might contribute to altered basal and inducible activities of the reporters. However, our findings are in agreement with Sturzenbecker et al.7 These authors did not find any differences in the ability of the VDR variants to transactivate a 24OHase-Luc reporter that contained nucleotides −644 to +74 of the rat 24OHase gene promoter.
Since the two VDR forms exhibited similar abilities to transactivate gene transcription from the 24OHase-Luc reporter, and because relative transactivation activities are potentially gene specific, we compared their activities with two other vitamin D responsive reporter genes. We found that the two receptor forms were equally active in transactivating OP-Luc and OC-Luc reporters that contain single VDREs. Similarly, Sturzenbecker et al.7 also found no differences in the transactivation activities between the receptor forms with an OC-Luc reporter, which contained three VDREs. Although our results show no differences in the functionality of the two receptor forms, we cannot rule out the possibility of a differential effect on other vitamin D responsive genes.
A transfected cell system is artificial and results in very high levels of VDR. This may potentially mask small differences in VDR function. We attempted to minimize this effect by decreasing the amount of VDR expression plasmid transfected. Even at very low levels of transfected DNA, we still found equivalent transactivation activities of the two VDR forms. Human fibroblasts, normally containing 20–50 fmol/mg of protein, may provide a more physiologically relevant model of 1,25(OH)2D3 action than transfected cells. However, as with our findings in transfected cells, in cultured normal fibroblasts, we found that induction of endogenous 24OHase mRNA in 1,25(OH)2D3 dose-response experiments was similar among cells with ff, Ff, and FF genotypes.
The ability to detect small differences in VDR transactivation activity in transfected cells and in normal cells is limited by the sensitivity of the assay methods used. In transfection experiments, we would have been unable to detect differences <15–20%. Northern analysis is somewhat less sensitive, and we estimate that we would not have detected less than ∼2-fold difference in 24-hydroxylase mRNA induction in fibroblasts. It is therefore possible that small yet physiologically important differences in VDR-mediated transactivation might have gone undetected. Moreover, we cannot exclude a cell-specific effect that might occur in tissues critical to skeletal and mineral homeostasis, such as bone and intestine. It is interesting to speculate that over the lifetime of an individual, the cumulative effect of cell-specific and very small differences in VDR function might contribute to differences in BMD. Furthermore, environmental factors such as dietary calcium and estrogen status might accentuate small differences in VDR function and thus lead to measurable differences in BMD across SCP genotypes.
In contrast to other members of the steroid-thyroid-retinoid receptor family, the VDR has a very short amino terminus. The A/B domain of the VDR, which encompasses amino acids 1–24 and includes the SCP, does not include a transactivation function (AF-1).27,28 Thus, the SCP may result in shortening of a region of the VDR that plays a negligible or very small role in determining VDR function. Since the SCP is associated with differences in BMD, yet does not appear to result in measurable changes in VDR function, one must consider alternate explanations for its mechanism. One possibility is that the SCP may be a marker for a nearby gene or genes that contribute to the genetic determination of BMD.
Our investigation of receptor function including ligand binding, VDR-DNA interaction, and transactivation of three vitamin D responsive genes did not detect any differences between the two VDR forms coded for by alternate SCP alleles. In conclusion, based on our extensive analysis, we are unable to define a molecular mechanism whereby the SCP might lead to differences in VDR function and as a result cause alterations in BMD. However, with currently available methods, we cannot rule out small differences between f-VDR and F-VDR function.
We thank Dr. W.J. Pike for providing the OC-Luc and OP-Luc reporters as well as the VDR antibody. We also thank Dr. H. DeLuca for the 24OHase-Luc reporter, Dr. K. Okuda for the rat 24-hydroxylase cDNA and Dr. Walter Horton for human dermal fibroblasts. This work was supported by National Institutes of Health grants DK-50802 (D.F.) and DK-02459 (C.G.).
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